Plasticity in Gene Expression in Response to Embryonic Environment Supporting Information

Anthony A Snead1, Corey R Quackenbush2, Shawn Trojahn2, Anna McDonald2, Luana Lins3 Chris Cornelius1, Paula E Adams4, Dengke Ma5 Yuying Hsu6, Eric Haag7, Frédéric Silvestre8, Akira Kanamori9, Ryan L Earley1*, Joanna L Kelley2*


Affiliations:
1Department of Biological Sciences, University of Alabama, 300 Hackberry Lane, Box 870344, Tuscaloosa, AL 35487
2School of Biological Sciences, Washington State University, 100 Dairy Road, Pullman, WA 99164
3Australian National Insect Collection, CSIRO, Canberra, Australia
4Department of Biological Sciences, Auburn University, Auburn, AL, USA
5Cardiovascular Research Institute and Department of Physiology, University of California San Francisco, San Francisco, California, USA
6Department of Life Sciences, National Taiwan Normal University, Taipei 116, Taiwan
7Department of Biology and Biological Sciences Graduate Program, University of Maryland, College Park, MD 20742
8Laboratory of Evolutionary and Adaptive Physiology, Institute of Life, Earth, and the Environment, University of Namur, 61 Rue de Bruxelles, 5000, Namur, Belgium
9Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Chūbu, Japan


Authors for correspondence:
*Joanna L. Kelley, School of Biological Sciences, Washington State University, 100 Dairy Road, Pullman, WA 99164, USA, 509-335-0037 (phone), 509-335-4848 (fax),
*Ryan Earley, Department of Biological Sciences, University of Alabama, 300 Hackberry Lane, Box 870344, Tuscaloosa, AL 35487


Developmental Formula

The formula to estimate when embryos should be removed from treatment was derived from a non-linear regression with “developmental stage (DS)” (1-35, from Harrington 1968) as the predictor and “hours of development” (0-310, from Mourabit et al. 2011) as the response. The resulting formula was:


Hours of Development ~ -33.04 + 4.37*DS - 0.07*DS2 - 0.007*DS3 + 0.0017*DS4 + 0.00009*DS5

Upon collecting the egg from its mother, we scored developmental stage (DS) and, using the above formula, calculated the estimated number of hours that the embryo had already spent developing and thus, how long it should remain in treatment before being sampled. We subtracted that value from 120 (with 120 hours being the time it takes the animal to develop to stage 29), 140 hours (stage 30), 180 hours (stage 31), 211 hours (stage 32), 240 hours (stage 33), or 310 hours (stage 34) (Mourabit et al. 2011). This value then was converted to days, and dictated when we sampled the animals from treatment. We confirmed DS under a stereomicroscope when the embryo was removed from treatment, and it was this confirmed DS that was used to determine whether the embryo was pre- or post-thermolabile period.


References

Harrington, Jr RW (1968) Delimitation of the thermolabile phenocritical period of sex determination and differentiation in the ontogeny of the normally hermaphroditic fish Rivulus marmoratus Poey. Physiological Zoology 41: 447-460.

Mourabit S, Edenbrow M, Croft DP, Kudoh T (2011). Embryonic development of the self-fertilizing mangrove killifish Kryptolebias marmoratus. Developmental Dynamics 240: 1694-1704.



Supporting Figures


SI Figure 1 The graph visualizing the non-linear regression with developmental stage, shortened to “DS” for the equation, as the predictor (x-axis) and the hours of development as the response (y-axis). The points are data used to derive the formula, and the line is the non-linear regression. The equation is displayed in the upper right corner of the graph for reference.



SI Figure 2 The multidimensional scaling plot for the 500 genes with the largest fold change between samples using the first two principal components. Each group (Pre-Cold, Pre-Warm, Post-Cold, and Post-Warm) is differentiated by shape and color in the legend.



SI Figure 3 The multidimensional scaling plot for the 1000 genes with the largest fold change between samples using the first two principal components. Each group (Pre-Cold, Pre-Warm, Post-Cold, and Post-Warm) is differentiated by shape and color in the legend.



SI Figure 4 The multidimensional scaling plot for the 10000 genes with the largest fold change between samples using the first two principal components. Each group (Pre-Cold, Pre-Warm, Post-Cold, and Post-Warm) is differentiated by shape and color in the legend.


SI Figure 5 The four-panel interactive plot is faceted by the comparison (Post-Cold vs. Post-Warm, Pre-Cold vs. Post-Cold, Pre-Cold vs. Pre-Warm, Pre-Warm vs. Post-Warm). Each point is a gene, and each panel has the negative log10 of the false discover rate adjust p-value (log odds probability) on the y- axis and the log fold change on the y axis. The dashed grey line is the significance cut off (FDR < 0.05); therefore, any genes above the line are significantly differentially expressed between the treatments.

Supporting Tables

SI Table 1 The interactive table includes all the temperature measurements for each treatment ( Warm = 25°C, Cold = 20°C) for all dates. The date column contains the date (Year-Month-Day) and time (Hour:Minutes:Seconds). Drastic changes in temperature correspond with the retrieval of the temperature probes to download data.



SI Table 2 The table contains read information per sample. Each sample is identified their sample name along with the number of reads after trimming, subsampling, and the percentage of the subsampled reads that were mapped to a gene.

Sample Name Trimmed Subsampled Percent Mapped (%)
RPRW_112 8,326,750 8,326,750 42.50
RPOC_250 8,158,694 8,158,694 85.97
RPOC_255 7,454,576 7,454,576 83.69
RPOC_259 7,176,142 7,176,142 84.10
RPRW_272 14,121,180 14,121,180 45.82
RPOW_283 8,788,552 8,788,552 84.39
RPOW_285 10,008,752 10,008,752 90.14
RPRC_28 60,088,608 40,000,000 90.83
RPRC_38 50,075,722 40,000,000 90.09
RPRW_39 49,014,042 40,000,000 91.60
RPOW_58 9,886,540 9,886,540 83.99
RPRC_9 11,685,830 11,685,830 83.95




SI Table 3. The interactive table contains all the differential gene expression results. Each row contains the gene being analyzed, the log fold change, the log Counts per Million (CPM), the false discovery corrected p value (FDR), and the comparison (Post-Cold vs. Post-Warm, Pre-Cold vs. Post-Cold, Pre-Cold vs. Pre-Warm, Pre-Warm vs. Post-Warm).



SI Table 4 The interactive table includes the fisher’s exact test gene ontology enrichment results with the comparison, false discovery rate corrected p. value (FDR), term ID, Source, and term name. Term ID refers to the numerical code for the term name, while term name is the gene ontology. Source is the gene ontology the term belongs too (Biological Process, Molecular Function, Cellular Component).